Addressing the Insufficiency of Marine Outfall Regulations in Mitigating Microplastic and AMR Pollution from Wastewater Treatment

<title>Abstract</title> Microplastics are found in all marine compartments, ranging from sea ice to sediments, beaches, and the open ocean, with a significant portion originating from land-based wastewater treatment plants. Policy in the South African context permits responsible disposal of wastewater through a marine outfall provided that the raw wastewater undergoes treatment and does not cause any adverse effects to the receiving body. However, a desktop study focused on regulatory framework for marine outfalls in South African coastal cities shows that partially treated (primary or preliminary treated) wastewater could be deposited into oceans. Microplastics form a considerable proportion of the wastewater mixture but also Enterobacteriaceae. To demonstrate the relevance of the wastewater treatment plant (WWTP) bacteria in biofilms on microplastics, this study aimed at identifying and characterizing Enterobacteriaceaeisolates from a simulated marine environment using microcosms. Experiments challenging the regulations were based on microbiology of the effluent that is discharged through an outfall. Microcosms were set up by spiking seawater with WWTP effluent and adding the sterilized collected plastic pieces. Scanning electron microscopy (SEM) was used to determine colonization on the microplastics. After 30 days of microcosm exposure, selective media and incubation conditions were used to isolate Enterobacteriaceae. Pure isolates were tested against 16 antibiotics normally used in human clinical settings. In the initial biofilms directly from microplastics from the WWTPs, several genera generally associated with wastewater treatment were isolated. Dominant species isolated and identified were <italic>Citrobacter </italic>sp., <italic>Escherichia </italic>sp., <italic>Enterobacte</italic>r sp., <italic>Serratia </italic>sp., <italic>Klebsiella </italic>sp. and <italic>Pseudomonas </italic>sp.. Several isolates were resistant to carbapenems (doripenem and imipenem; 9% to 27%). Some isolates were resistant to up to ten of the antibiotics. Bacterial infections caused by Carbapenem-resistant Enterobacteriaceae have become a global concern in the fight against bacterial infections. The results of the present project show that clinically relevant Enterobacteriaceae colonize microplastics and that these survive in biofilms on these microplastics surfaces. Such results suggest that more data is needed that will challenge the current policy regulating the release of sewage through marine outfalls as well as the notion that dilution of sewage is an answer to pollution.